It has been well documented in the art that many diverse biological functions are associated with the presence of sialoglycoproteins and sialoglycolipids at or near the cell surfaces in mammals. It has been shown, for example, that the metastasis of malignant cells may be a direct function of an excessive release by the malignant cells, or by other cells, of sialoproteins or sialopeptides into the blood stream. The following is a list of representative literature references documenting the correlation between various biological functions and the presence of at or near the cell surface or the release from the cell of sialopeptides or sialoproteins: Gandhi et al, Effect of Diabetes Mellitus on Sialic Acid and Glutathione Content of Human Erythrocytes of Different Ages, Indian Journal of Experimental Biology, Vol. 17, pp. 585-587 (June, 1979); Wautier et al, A Platelet Defect in a Patient with Eosinophilic Leukaemia: Ristocetin-Induced Platelet Aggregation Associated with a Reduced Platelet Sialic Acid Content, Scand. J. Haematol., Vol. 22, pp. 267-276 (1979); Sato et al, Target of X Irradiation and Dislocation of Sialic Acid in Decrease of Cell Surface Charge of Erythrocytes, Radiation Research, Vol. 69, pp. 367-374 (1977); Buck et al, Sialoglycoprotein Differences Between Xenotransplantable and Nonxenotransplantable Ascites Sublines of the 13762 Rat Mammary Adenocarcinoma, Archives of Biochemistry and Biophysics, Vol. 198, pp. 12-21 (1979); Dawson et al, Variations in Sialomucins in the Mucosa of the Large Intestine In Malignancy: A Quantative and Statistical Analysis, Biochemical Journal, Vol. 10, p. 559 (1978); Hakim, Correlation Between Perchloric-Acid-Soluble Serum Proteins, Cellular Immunity and Tumor-Cell Burden, Int. J. Cancer, Vol. 25, pp. 281-288 (1981); Hassing et al, Ultraviolet Difference Spectral Studies on Concanavalin A-Carbohydrate Interaction, Eur. J. Biochem. Vol. 16, pp. 549-556 (1970); Lipton et al, Glycoproteins and Human Cancer, Cancer, Vol. 43, pp. 1766-1771 (1979); Gorman et al, Determination of Protein-Ligand Equilibria by Difference Spectroscopy. Hemerythrin-Ligand Thermodynamic Studies, Biochemistry, Vol. 20, pp. 38-43 (1981); Moss et al, Significance of Protein-Bound Neuraminic Acid Levels in Patients with Protein-Bound Neuraminic Acid Levels in Patients with Prostatic and Bladder Carcinoma, Urology, Vol. 13, p. 182 (1979); Codington et al, Variations in the Sialic Acid Compositions in Glycoproteins of Mouse Ascites Tumor Cell Surfaces, Biochemistry, Vol. 18, pp. 2145-2149 (1979); Sherblom et al, Purification of the Major Sialoglycoproteins of 13762 MAT-B1 and MAT-C1 Rat Ascites Mammary Adenocarcinoma Cells by Density Gradient Centrifugation in Cesium Chloride and Guanidine Hydrochloride, The Journal of Biological Chemistry, Vol. 255, pp. 783-790 (1980); Jumblatt et al, Altered Surface Glycoproteins in Melanoma Cell Variants With Reduced Metastasizing Capacity Selected for Resistance to Wheat Germ Agglutinin, Biochemical and Biophysical Research Communications, Vol. 95, pp. 111-117 (1980); Vilarem et al, Differences in Sialic Acid Contents of Low Cancer Cells, High Cancer Cells and Normal Mouse Lung Counterparts, Biochemical and Biophysical Research Communications, Vol. 98, pp. 7-14 (1981); Gaffar et al, Further Studies on a Human Lung Tumor-associated Antigen, The Journal of Biological Chemistry, Vol. 254, pp. 2097-2102 (1979); Blithe et al, Comparison of Glycopeptides from Control and Virus-Transformed Baby Hamster Kidney Fibroblasts, Biochemistry, Vol. 19, pp. 3386-3395 (1980); Santer et al, Partial Structure of a Membrane Glycopeptide from Virus-Transformed Hamster Cells, Biochemistry, Vol. 18, pp. 2533-2540 (1979); Yogeeswaran et al, Cell Surface Sialic Acid Expression of Lectin-Resistant Variant Clones of B16 Melanoma With Altered Metastasizing Potential, Biochemical and Biophysical Research Communications, Vol. 95, pp. 1452-1460 (1980); Glick, Membrane Glycopeptides from Virus-Transformed Hamster Fibroblasts and the Normal Counterpart, Biochemistry, Vol. 18, p. 2525 (1979); Allen et al, Glycoprotein Receptors for Concanavalin A Isolated from Pig Lymphocyte Plasma Membrane by Affinity Chromatography in Sodium Deoxycholate; Nature New Biology, Vol. 236, pp. 23-25 (1972); Hayman et al, Purification of Virus Glycoproteins by Affinity Chromatography Using Lens Culinaris Phytohaemagglutin, Febs, Letters. Vol. 29, pp. 185-188 (1973) Lotan et al, Activities of Lectin and Their Immobolized Derivatives in Detergernt Solutions. Implications on the Use of Lectin Affinity Chromatography for the Purification of Membrane Glycoproteins, Biochemistry, Vol. 16, pp. 1787-1794 (1977).
Lectins are a group of proteins that often demonstrate high binding specificity toward carbohydrate residues of glycoproteins and glycolipids. They have proven invaluable in the study of glycoproteins of cell surfaces as well as the modifications they undergo during cell differentiation and malignant transformation. Relatively few lectins with specificity for sialic acid have been identified. Two such lectins, limulin and carcinoscorpin, have been purified from the American horseshoe crab, Limulus polyphemus, and the Indian horseshoe crab, Carcinoscorpius rotunda cauda, respectively. Limulin also binds N-acetylglucosamine and D-glucuronic acid. The carbohydrate specificity of carcinoscorpin has not been extensively studied. A lectin, LAg-1, which binds N-acetylneuraminic acid, N-glycoylneuraminic acid and N-acetylmannosamine has been isolated from the lobster, Homarus americanus. A lectin which binds sialic acid residues of glycoproteins has also been isolated from wheat germ. Agglutination of erythrocytes by wheat germ agglutinin is inhibited by N-acetyl-D-galactosamine and N-acetylneuraminic acid, with N-acetyl-D-glucosamine being the more potent inhibitor.
It has been reported that extracts of the slug, Limax flavus, agglutinate red blood cells (Pemberton, Vox Sang., Vol. 18, ppl 74-76 (1970); however, no carbohydrate specificity was defined for the agglutinin.
The specific sialoprotein binding lectin (limulin and carcinoscorpin) of the prior art are relatively unstable, however, due to their large molecular size and multisubunit composition (18 to 20). Moreover, due to the scarcity of the sources for the lectins, the latter are expensive to prepare or procure.
It is an object of the present invention to provide a highly specific sialic acid binding lectin.
It is a further object of the present invention to provide methods for obtaining homogeneous preparations of this lectin from the slugs, Limax flavus.
It is a further object of the present invention to provide methods for quantitating the amount of sialoproteins or sialopeptides present in biological fluids or cellular membranes.
It is a further object of the present invention to provide and/or describe methods for the separation and comparison of the sialoproteins and sialopeptides present in biological fluids or solubilized membrane proteins.
It is a further object of the present invention to provide methods for the purification of sialoproteins and sialopeptides in quantities sufficient for antibody preparation to said sialoproteins or sialopeptides.